The present invention relates to a high capacity binary shuttle vector for plant transformation, etc. In particular, the present invention relates to a high capacity binary shuttle vector which is used for seed plants transformation, particularly monocotyledonous plants which were difficult to transform in spite of their economical importance, but it is also used very powerfully for the complementation test analysis of genome functions and the construction of a genome library for genome function analysis.
Monocotyledonous plants are more difficult to transform than dicotyledonous plants, in spite of their importance in agriculture. The conventional transformation of these plants has been carried out by various forced introduction methods such as those using gene guns for intact cells, or electroporation or PEG (polyethylene glycol) method for protoplasts.
However, there are problems in the electroporation method or PEG method that regeneration from protoplasts to normal plants is difficult, and in case of gene gun, that expensive facilities are required and a large number of samples can not be processed simultaneously. Furthermore, a large gene fragment of 10 kb or more into monocotyledonous plants has not been introduced without its rearrangement.
Recently, Hiei et al. have reported that rice, a typical monocotyledonous crop can be transformed with a pBI system vector and Agrobacterium (Hiei et al., Plant Journal, 6:271-282 (1994)). This has made the transformation of rice drastically easy.
However, the pBI vector used by Hiei et al. can stably introduce only about 10 kb DNA into rice. If larger DNA was integrated into such a shuttle vector, the vector could not be stably maintained even in E. coli. 
By the recent advances of genome studies, the positional cloning method to isolate a gene, which is based on its positional information on the chromosome map, became the focus of interest as a promising method to isolate the useful gene determining agricultural traits. In order to apply this technique to identify genes causing these traits from the candidate genes selected by using their map information, the trait expression by the gene in the genome fragment must be confirmed by transformation of the fragment to plant. In this case, introduction of the larger genome fragment makes the screening of genes the more efficient. Therefore, it is desirable to introduce as large a genome fragment as possible.
Although the binary cosmid method and PEG method are known to introduce genome fragments of 10 kb or larger size into a plant, both of the methods have deficiencies as follows:
(1) As for the binary cosmid method, the upper size of a genome fragment to be introduced is limited up to about 20 kb, and pBI system RK2 is used as replication origin. Therefore, it has been often difficult to stably maintain the plasmid even in E. coli into which the plasmid with the chromosome fragment is to be introduced at first.
(2) As for the PEG method, the regeneration from protoplasts requires high proficiency of the skill, and often the introduced fragments are severely rearranged making normal complementation by the fragment rather difficult. Therefore, such methods are not sufficiently suited to assay the functions of large genome fragments.
The present invention was made in view of the circumstances described above. The object of the present invention is to introduce a genomic DNA fragment of 10 kb or larger size into plants, particularly higher plants. And it can be also used to monocotyledonous plants which are difficult to transform although many of them are agriculturally important.
Another objective of the present invention is to establish an efficient and stable complementation analysis method for a genome region of 10 kb or larger than that, which is very difficult to be introduced into plants by conventional methods. By introducing such a large genome fragment efficiently to plants, isolation of the genes controlling various agricultural traits can be more efficient. Another object of this invention is to introduce a large gene complex into a plant as a block to enable them to express complicated traits in plants. Sometimes such gene complex is located in a group as an operon.
That is, the present invention relates to a high capacity binary shuttle vector comprising T-DNA region, and Ri replication origin.
The high capacity binary shuttle vector of the present invention is a binary shuttle vector.
Further, said high capacity shuttle vector comprises a gene with a multi-cloning site in the T-DNA region, specifically lacZ gene.
Further, said high capacity binary shuttle vector comprises some or an antibiotic resistant genes being introduced between the lacZ gene introduced in the T-DNA region and the border regions of the T-DNA. Said border region is preferably a left border region, and said antibiotic resistance gene is preferably a hygromycin resistant gene.
Another selection marker antibiotic resistance can be kanamycin resistance gene which can be used as a selection marker in E. coli, and dicotyledonous plants.
Further, said high capacity shuttle vector may integrate a fragment with various sizes ranging from a small DNA fragment to a large genome fragment in it. It is confirmed that the genome fragments sized up to at least 40 kb can be efficiently integrated to rice, and larger size of it may be possible to be integrated.
The present invention is a binary vector comprising a lox site in T-DNA region, a par C gene and an Ri ori as replication origin. The vector is capable to integrate a clone plasmid of a circular genome library with lox site by cre enzyme, in a host, E. coli or Agrobacterium.
The present invention is also a binary vector for plant transformation comprising multi-cloning site sandwiched by with a promoter and a terminator for plants in a T-DNA region, and an Ri ori as replication origin.
The present invention further is a genomic library having the ability to transform plants. Each clone constituting the genomic library having the ability to transform plants has an insert in the T-DNA region of the high capacity binary shuttle vector of the present invention.
The present invention is complementation method for assessing the function of genes in a genome fragment inserted in a clone of a genome library. This method comprises introducing said plasmid with a genome insert into an Agrobacterium cell, and transferring said plasmid into a plant. Furthermore, the present invention is another complementation method for assessing the function of genes in a genome fragment inserted in a clone of a genome library. This method comprises integrating a component plasmid of a library with a genome insert and a binary vector as described in claim 8, wherein said library is constructed by using a circular vector with lox site and has E. coli as a host, introducing said integrated vector into an Agrobacterium cell, and then transferring said integrated vector into a plant.
The present invention is a gene obtained with the complementation method described above.
The present invention is a screening method for a useful gene by using the above-mentioned high capacity binary shuttle vector or the library integrating vector.
Furthermore, the present invention is a transformed plant with said high capacity binary shuttle vector. The plant to be transformed includes all kinds of plants, mainly seed plants, and particularly monocotyledonous plants.
The present invention is a method for producing a high efficiency Agrobacterium strain with recAxe2x88x92 to maintain stably the binary vector with a large insert. This method comprises generating high transformation efficiency recAxe2x88x92 strains by the site-directed mutation on recA gene of said strain to transform to a recAxe2x88x92 strain, introducing a homologous recombination to the strains with recA+ genes, between the transformed recAxe2x88x92 vector and recA+ gene in the cell, selecting the recombinant by making replica of the plate on which the strains are spread, and irradiating the plate to screen a clone which can not grow under UV irradiation. Or such recAxe2x88x92 strain can be selected by replica after normal mutagenesis and selection under UV irradiation.
The present invention is a high transformation efficiency Agrobacterium strain with recAxe2x88x92, wherein said strain can maintain stably the said high capacity binary vector with large insert, and can transform various normally recultitrait plants for transformation.